High performance liquid chromatography (HPLC) is a technique used to separate compounds in a liquid mixture. An HPLC system consists of a solvent reservoir, pump, injector, separation column, and detector. The mixture is injected and the components separate as they pass through the column at different rates depending on how they partition between the mobile and stationary phases. HPLC is used for research, quality control, and regulatory purposes to separate, analyze, purify, and quantify complex mixtures like drugs, metabolites, polymers, proteins, lipids, and environmental pollutants.

1. S.N.PATEL INSTITUTE OF
TECHNOLOGY AND RESEARCH
CENTRE,UMRAKH
HPLC(High Pressure Liquid Chromatography)
Subject : P& IC(2140501)
Guided by: Mr. Anish Solanki

2. High Performance
Liquid
Chromatography

3. Principle
 The principle of this technique is that liquid
mixture passing by the moving liquid phase(as
mobile phase) over solid column (as Stationary
phase) having small diameter under pressure,
its constituents are separated, which are
detected using detector and gives its identical
chromatograph peak.

4. COMPOSITION OF A LIQUID
CHROMATOGRAPH SYSTEM
 Solvent Reservoir
 Solvent Delivery System (Pump)
 Injector
 Sample
 Column
 Detectors (Diode Array)
 Waste Collector
 Recorder (Data Collection)

5. Introduction
 HPLC is a form of liquid chromatography used
to separate compounds that are dissolved in
solution. HPLC instruments consist of a
reservoir of mobile phase, a pump, an injector,
a separation column, and a detector.
 Compounds are separated by injecting a
sample mixture onto the column. The different
component in the mixture pass through the
column at differentiates due to differences in
their partition behavior between the mobile
phase and the stationary phase. The mobile
phase must be degassed to eliminate the
formation of air bubbles.

6. HPLC system

8. Picture of HPLC instrument

9. Uses of HPLC
 This technique is used for chemistry and biochemistry
research analyzing complex mixtures, purifying
chemical compounds, developing processes for
synthesizing chemical compounds, isolating natural
products, or predicting physical properties. It is also
used in quality control to ensure the purity of raw
materials, to control and improve process yields, to
quantify assays of final products, or to evaluate product
stability and monitor degradation.
 In addition, it is used for analyzing air and water
pollutants, for monitoring materials that may jeopardize
occupational safety or health, and for monitoring
pesticide levels in the environment. Federal and state
regulatory agencies use HPLC to survey food and drug
products, for identifying confiscated narcotics or to
check for adherence to label claims.

10. Uses of HPLC
 Quantitative Analysis: As large number of compounds
have tendency to absorbed in column, so it is most
widely used to separate and detect the compounds from
the mixture.
 HPLC is widely used to separate the polar compounds
like drugs and their metabolism ,peptides, vitamins,
amino acids, polyphenols and steroids.
 Molecular weight of polymers is determined using
HPLC combination.
 Various types of drugs,lipids, proteins, amino acids,
carbohydrates have been analyzed by HPLC.